1. Field of the Invention
This invention relates to the isolation and purification of taxane derivatives from a naturally occurring Taxus species, and more particularly, to an improved method for isolating taxol and the congeners thereof from the Taxus species by reverse phase liquid chromatography.
2. Related Art
Taxol was first isolated in 1971 from the western yew, Taxus brevifolia by Wani, et al. (J. Am. Chem. Soc., 93:2325, 1971), who characterized its structure by chemical and X-ray crystallographic methods.
Taxol is a member of the taxane family of diterpenes having the following structure: ##STR1##
Taxol and various taxane derivatives including cephalomannine are highly cytotoxic and possess strong in vivo activities in a number of leukemic and tumor systems. Especially, taxol is considered an exceptionally promising cancer chemotherapeutic agent, and is currently in phase II clinical trials in the United States. However, the major problem with the ongoing clinical trial is the limited availability of the compound.
The only available natural source for taxol so far is several species of very slowgrowing yew (genus Taxus, family Taxaceae). The isolation procedures currently practiced are very difficult, low-yielding, and obviously fatal to the source. For example, C. H. O. Huang et al. (J. Nat. Prod., 49, 665, 1986) reported a 0.01% yield from a large scale isolation starting with 806 lbs. or more of Taxus brevifolia bark. According to Huang's method, the ground bark is extracted with methanol. The methanol extract is concentrated and the concentrate extracted with dichloromethane. The dichloromethane extract is then concentrated and the concentrate is dispersed in 1:1 acetone-hexane. The soluble portion is applied to a column of Florisil in hexane/acetone and then eluted with hexane containing an increasing amount of acetone (30%-50%). The taxol-containing fractions are collected and concentrated to obtain a crude composition containing taxol. The taxol in the crude composition is recrystallized once from methanol-water and a second time from hexane-acetone. The recrystallized taxol is further purified by chromatography on silica gel and recrystallized again. All the mother liquors are combined, recrystallized, and chromatographed to yield additional quantities of purified taxol. The isolation of taxol was described by other workers: R. W. Miller, et al., J. Org. Chem., 46, 1469, 1981; J. L. McLaughlin, et al., J. Nat. Prod., 44, 312, 1981; D. G. I. Kingston, et al., J. Nat. Prod., 45, 466, 1982; and V. Senih, et al., J. Nat. Prod., 47, 131, 194. The reported yields of taxol from various species of yew range from 50 mg/kg to 165 mg/kg (i.e., 0.005-0.017%).
In view of the high therapeutic potential of taxol, the synthesis of the compound has attracted much interest among synthetic organic chemists worldwide. Disappointedly, the synthesis of a natural product with such complexity, however, appears to be of little practical value.
Because of the current state of synthetic efforts, the isolation of taxol from Taxus species, despite low yields, will be the only reliable supply source for clinical quantities of taxol for years to come. Thus, purification techniques which provide higher yields of taxol are needed to provide greater quantities of this promising therapeutic agent. The present invention provides a purification technique which accomplishes this goal.